MicroRNA-138 And MicroRNA-346 Co-regulates The Growth Of Human Cervical Cancer HeLa Cells By Targeting HTERT

Aims:The occurrence and development of tumor are cascade of linked sequential steps involving multiple regulated genes,the crypticity and predictability are the most insidious and life-threatening aspects of cancer.Our understanding of the processes of tumor has improved,and the activation of telomerase was an main step of cancer.(miRNAs)are a class of small non-coding RNAs that post-transcriptionally regulate gene expression.Recent evidences indicate that special miRNAs may function as tumor suppressors or oncogenes and play a critical role in cancer initiation and progression by negatively regulating their target genes.This article aims to illustrate the function of miRNAs regulate hTERT,which has a high expression in cervical cancer and maintain the malign hyperplasia of tumor,and explore the mechanism of various kinds of miRNAs target the single gene.Methods:We detected the differencial expression of miR-138 in human cervical cancer and adjacent normal tissues by real-time RT-PCR assay.Both level and function of miR-138 were enhanced in human cervical tumor cell line HeLa and the changes of cell phenotypes were detected with MTT assay,colony formation assay.Subsequently,we used bioinformation and identified the candidate target genes for miR-138.The reliability of the direct target gene was confirmed by fluorescent reporter experiment.Furthermore,the mRNA levels and protein levels of target gene in miR-138-enhanced cervical cancer cells or tissues were detected with real-time RT-PCR and western blot,in order to confirm the regulating role of miR-138 in target gene expression.Finally,the function of miR-138 was rescued by epotic overexpression hTERT without 3'UTR in human cervical tumor cell line HeLa.And then we use EGFP report assay and Western Bolt assay to confirm the relationship of miR-346 and miR-138,which has the overlap binding sites in hTERT-3'UTR.Results:Experimental results showed that the expression of miR-138 in human human cervical tumor tissues was lower than in adjacent normal tissues.We found that after overespression of miR-138,the HeLa cell proliferation activity and the colony formation activity were both inhibited.Subsequently,we identified tumor related gene Human Telomerase Reverse Transcriptase(hTERT)as one of the candidate target genes for miR-138.The hTERT mRNA 3'-untranslated region(3'UTR)contains the potential binding site of miR-138.The fluorescent reporter experiment also confirmed that miR-138 can directly bind to the hTERT mRNA 3'UTR and negatively regulate the gene expression.After overexpression of miR-138 in cervical tumor cells or tissues,protein level of hTERT was descended.Finally,we found that overexpresion of hTERT without 3'UTR can rescued the HeLa cell proliferation activity and the colony formation activity,which was inhabited by miR-138.At last,we demonstrate that regulations of miR-346 and miR-138 on hTERT expression are opposite through the competitive binding its 3'UTR,and hTERT expression is directly correlated with the ratios of miR346 to miR-138 levels.Conclusions:Our results indicate that in Human cervical tumor,miR-138 functions as a tumor suppressor gene and inhibites cell proliferation.The ability of downexpressed miR-138 in HeLa cells directly down-regulating the expression level of tumor related gene hTERT is decreased and result in the enhancement-of-function of hTERT,leading to the activation of cell proliferation.The elucidation of common mechanisms of miR-138 in human cervical tumor helps us to further understand cancer initiation and progression,and provides new evidences to cancer diagnosis and therapy.Based on the current issues,we found that two miRNAs can coordinately regulate the expression of a single targeting mRNA,which provide a new insight into mechanism of miRNA regulating mRNA.